PROJECT SUMMARY OBJECTIVE: We will determine the molecular mechanism by which the aqueous humor outflow (AHO) directs the formation of Schlemm?s canal (SC) through Klf4. SC is a specialized vascular structure that drains the aqueous humor from the anterior chamber into the circulation, and plays a key role in regulating the intraocular pressure (IOP). Dysfunctional SC due to aging or diseases could critically elevate the IOP and often causes ocular nerve damage, possibly leading to glaucoma. Better understanding of the reciprocal interaction between SC development and the AHO would thus have a transformative impact on the prevention of glaucoma caused by elevated IOP. RATIONALE: Structurally, SC is directly connected to the aqueous vein to drain the aqueous humor. Because of this direct vascular joining, SC has long been thought to be a specialized venous extension, whose inner wall is lined by blood vascular endothelial cells. Interestingly, however, several studies have demonstrated evidences, which distinguish SC from typical blood vessels and re-categorize SC as a new lymphatic-like vascular structure. These studies have prompted us to carefully re-examine the molecular and cellular features of SC. As the results, we and others have recently uncovered that SC is postnatally derived from the limbal vascular plexus by upregulating Prox1, the master regulator of lymphatic development. Importantly, this lymphatic reprograming of blood vessel endothelial cells (BECs)-to-SC endothelial cells (SCECs) appeared to be triggered and maintained by the optimal AHO. Accordingly, our main question to address here is how the mechanical signal from the AHO regulates the genetic program that specifies the SCEC identity. STRATEGY & GOAL: In addition to our lymphatic-specific fluorescent mouse model that was published recently, we have created a transgenic rat model whose lymphatic vessels and SC are genetically labeled with GFP. From these two novel murine models, we will purify and culture SCECs in vitro for various molecular and cellular characterizations of rodent SCECs. We have recently reported that Klf4, a shear stress responsive transcription regulator, is highly expressed in the SC precursor cells, and that Klf4 physically interacts with Prox1. Accordingly, we hypothesize that the fluid flow-induced mechanical signal may be incorporated via Klf4 into Prox1-mediated cell fate specification program, which together controls differentiation of limbal blood vessel BECs to SCECs. In Aim1, we will elucidate the mechanism of flow-mediated SCEC-fate specification through Klf4 using purified SCECs. As a preliminary study, we successfully isolated mouse SCECs and confirmed the presence of two unique ultrastructures of SCECs, namely giant vacuole and trans-cellular pores. We will study the roles of Klf4 in various molecular and cellular characteristics of isolated SCECs. In Aim2, we will employ two cohorts of tissue-specific, inducible Klf4 knockout mouse models to study the contribution of Klf4 to the initial stage of the SC organogenesis. Together, our proposed studies will provide a unique capability to address important unanswered questions on SC development, and generate valuable information to better understand the functional interaction between SC development and the IOP control with a possible therapeutic implication toward glaucoma.